Journal: Microarrays

Article Title: Evaluation of Solid Supports for Slide- and Well-Based Recombinant Antibody Microarrays

doi: 10.3390/microarrays5020016

Figure Lengend Snippet: Evaluation of slide-based solid supports for antibody microarrays. The supports were compared with respect to blocking buffer, surface fouling, spot morphology and signal strength. ( A ) The slides (no antibodies printed) were first blocked and then incubated with crude, labelled serum and scanned after washing. Any observed signal intensity represents non-specific background binding. Representative scans of FAST, black MaxiSorp, Nexterion H, epoxy polymer and NHS glass slides are shown. The blocking agents tested are shown to the right; ( B ) Scanned microarray images of 14 × 8 antibody microarrays on epoxy glass, epoxy polymer, NHS glass, Nexterion H, black MaxiSorp, GAPSII, Nexterion P, Silane-Prep and NHS polymer slides. The printed antibody arrays were blocked and then incubated with crude, labelled serum before scanning.

Article Snippet: For manual processing, individual sub-arrays were created by using silicon incubation chambers (Schott) or a hydrophobic pen (Dako, Glostrup, Denmark) for slides that did not fit the incubation chambers (Silane-Prep (Sigma) and GAPSII (Corning)).

Techniques: Blocking Assay, Incubation, Binding Assay, Polymer, Microarray

Journal: Cell Communication and Signaling : CCS

Article Title: Dysregulated Gab1 signalling in triple negative breast cancer

doi: 10.1186/s12964-024-01542-9

Figure Lengend Snippet: Constitutive signalling in breast cancer cell lines. A MCF7, T-47D, SKBR3, MDA-MB-468, UACC-3199, MDA-MB-231 and Hs 578T cells were seeded and cultivated in phenol red-free RPMI 1640 for 24 h. On the following day, cells were lysed and proteins were separated by SDS-PAGE. After Western blotting, membranes were stained with specific antibodies against vimentin, E-cadherin, (p)Y EGFR, EGFR, (p)Y Gab1, Gab1, (p)T/Y ERK1/2, ERK1/2, (p)S Akt, Akt, (p)Y STAT3, STAT3 and tubulin. A representative result of n = 3 independent experiments is shown. B Expression or phosphorylation of (p)Y EGFR, EGFR, (p)Y Gab1, Gab1, (p)T/Y ERK1/2, (p)S Akt and (p)Y STAT3 from n = 3 independent experiments were quantified and normalised to tubulin expression. The highest phosphorylation or expression among the cell lines analysed was set to 100%. High expression or phosphorylation is visualised in dark red. For each cell type the normalised phosphorylation and expression strengths of all analysed entities are summed up and depicted in blue. C MDA-MB-468 or MDA-MB-231 cells were seeded on poly-L-lysine-coated glass cover slips and cultivated in phenol red-free RPMI 1640. After 24 h, cells were transfected with an expression vector for murine Gab1-GFP. On the following day, cells were placed into the incubation chamber of a laser scanning microscope. Imaging was performed after 30 min equilibration. Representative results of n = 3 independent experiments are shown

Article Snippet: Prior to usage, the temperature of the incubation chamber (PeCon, Erbach, Germany) and of the objective lens was adjusted to 37 °C and the atmosphere within the incubation chamber was set to 5% CO 2 . eGFP-Gab1 fusion proteins were excited using laser light of 488 nm.

Techniques: SDS Page, Western Blot, Staining, Expressing, Phospho-proteomics, Transfection, Plasmid Preparation, Incubation, Laser-Scanning Microscopy, Imaging

Journal: Cell Communication and Signaling : CCS

Article Title: Dysregulated Gab1 signalling in triple negative breast cancer

doi: 10.1186/s12964-024-01542-9

Figure Lengend Snippet: EGFR activity promotes constitutive Gab1 and MAPK phosphorylation in MDA-MB-468 cells. A MDA-MB-468 cells were seeded and cultivated in phenol red-free RPMI 1640 for 24 h. On the following day, cells were treated with DMSO or Gefitinib (3 µM) for 30 min. Subsequently, cells were lysed and proteins were separated by SDS-PAGE. After Western blotting, membranes were stained with specific antibodies against (p)Y Gab1, Gab1, (p)T/Y ERK1/2, ERK1/2, (p)S Akt, Akt, (p)Y STAT3, STAT3 and tubulin. A representative result of n = 3 independent experiments is shown. The results from ( A ) were quantified. The diagrams show the ratios of B (p)Y Gab1 to Gab1, C (p)T/Y ERK1/2 to ERK1/2, D (p)S Akt to Akt, and E (p)Y STAT3 to STAT3. Gab1, ERK1/2, Akt and STAT3 phosphorylation in DMSO-treated cells was normalised to 100% in each independent repetition of the experiment. Data are given as mean of three independent experiments ± SD. Student’s t-test: n.s. = non-significant, * = p < 0.05, ** = p < 0.01, *** = p < 0.001. F MDA-MB-468 cells were seeded on poly-L-lysine-coated glass cover slips and cultivated in phenol red-free RPMI 1640. After 24 h, cells were transfected with an expression vector for murine Gab1-GFP. On the following day, cells were placed into the incubation chamber of a laser scanning microscope and left for 30 min. Cells were treated with Gefitinib (3 µM) or DMSO for 30 min. Imaging was performed after treatment with Gefitinib or DMSO. Representative results of n = 3 independent experiments are shown

Article Snippet: Prior to usage, the temperature of the incubation chamber (PeCon, Erbach, Germany) and of the objective lens was adjusted to 37 °C and the atmosphere within the incubation chamber was set to 5% CO 2 . eGFP-Gab1 fusion proteins were excited using laser light of 488 nm.

Techniques: Activity Assay, Phospho-proteomics, SDS Page, Western Blot, Staining, Transfection, Expressing, Plasmid Preparation, Incubation, Laser-Scanning Microscopy, Imaging

Journal: Cell Communication and Signaling : CCS

Article Title: Dysregulated Gab1 signalling in triple negative breast cancer

doi: 10.1186/s12964-024-01542-9

Figure Lengend Snippet: MAPK and PI3K signalling are crucial for Gab1 plasma membrane recruitment in MDA-MB-468 cells. A MDA-MB-468 cells were seeded on poly-L-lysine-coated glass cover slips and cultivated in phenol red-free RPMI 1640. After 24 h, cells were transfected with an expression vector for either murine Gab1-GFP, murine Gab1-S552A-GFP or murine Gab1-ΔPH-GFP. On the following day, cells were placed into the incubation chamber of a laser scanning microscope. Imaging was performed after 30 min equilibration. Representative results of n = 3 independent experiments are shown. B MDA-MB-468 cells were seeded on poly-L-lysine-coated glass cover slips and cultivated in phenol red-free RPMI 1640. After 24 h, cells were transfected with an expression vector for murine Gab1-GFP. On the following day, cells were placed into the incubation chamber of a laser scanning microscope and left for 30 min. Cells were treated with DMSO, U0126 (10 µM) and/or LY294002 (40 µM) for 30 min. Imaging was performed before and after treatment. Representative results of n = 3 independent experiments are shown

Article Snippet: Prior to usage, the temperature of the incubation chamber (PeCon, Erbach, Germany) and of the objective lens was adjusted to 37 °C and the atmosphere within the incubation chamber was set to 5% CO 2 . eGFP-Gab1 fusion proteins were excited using laser light of 488 nm.

Techniques: Clinical Proteomics, Membrane, Transfection, Expressing, Plasmid Preparation, Incubation, Laser-Scanning Microscopy, Imaging

Journal: Cell Communication and Signaling : CCS

Article Title: Dysregulated Gab1 signalling in triple negative breast cancer

doi: 10.1186/s12964-024-01542-9

Figure Lengend Snippet: Gab1 is localised in the cytoplasm in PI3K and MAPK inhibition resistant MDA-MB-468 cells. MDA-MB-468 cells were seeded on poly-L-lysine-coated glass cover slips and cultivated in phenol red-free RPMI 1640. After 24 h, cells were transfected with an expression vector for murine Gab1-GFP. On the following day, cells were treated with DMSO, U0126 (10 µM) or LY294002 (40 µM). After 48 h cells were placed into the incubation chamber of a laser scanning microscope. Imaging was performed after 30 min equilibration. Representative results of n = 3 independent experiments are shown

Article Snippet: Prior to usage, the temperature of the incubation chamber (PeCon, Erbach, Germany) and of the objective lens was adjusted to 37 °C and the atmosphere within the incubation chamber was set to 5% CO 2 . eGFP-Gab1 fusion proteins were excited using laser light of 488 nm.

Techniques: Inhibition, Transfection, Expressing, Plasmid Preparation, Incubation, Laser-Scanning Microscopy, Imaging